Polymerase variants for template-independent enzymatic nucleic acids synthesis and kit comprising the same

ABSTRACT

Provided herein relates to DNA polymerase variants and kits including the same, where the DNA polymerase variant has an improved function and activity of performing template-independent nucleic acids synthesis using canonical nucleotides and non-canonical nucleotide analogues in a thermotolerant manner.

CROSS REFERENCE

This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/249,819, filed on Sep. 29, 2021, the content thereof is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to B-family DNA polymerase variants and kits comprising the same for use particularly in the context of de novo enzymatic nucleic acid synthesis.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 211019US-sequence listing.XML, created on Jul. 12, 2022, which is 98 kb in size. The information in the electronic format of Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

Enzyme-based de novo nucleic acid synthesis is an emerging, non-toxic method to substitute for the decades-old, toxic chemical phosphoramidite-based nucleic acid synthesis.

All living organisms rely on nucleic acid polymerases to efficiently duplicate their DNA. Owing to their DNA duplication function, most nucleic acid polymerases require a template to direct synthesis and incorporation of nucleotides into a growing nucleic acid strand. The template-dependent manner of nucleic acid synthesis requires nucleic acid polymerase to associate with a primer-template DNA before the nucleotide can be added to the 3′-terminus of primer by the polymerase. To ensure a high-fidelity DNA synthesis, nucleic acid polymerases have evolved a robust nucleotide-selection mechanism to accurately choose and incorporate the correct nucleotide corresponding to its complementary templating base during the nucleic acid synthesis. The active-site pocket of nucleic acid polymerase is pre-arranged in a proper geometry for accommodating a correct and matched canonical nucleotide having a normal 3′-hydroxyl (3′-OH) group. Therefore, the elimination or substitution of the 3′-OH group with a bulky, chemical group on the nucleotide, such as 2′,3′-dideoxycleotide (ddNTP) and 3′-O-azidomethyl-dNTP, respectively, may significantly alter the nucleotide configuration within the active-site pocket of nucleic acid polymerase and reduce the nucleotide binding affinity and overall DNA synthesis efficiency of nucleic acid polymerase. Likewise, any modifications on the nucleobase or 5′-triphosphate group of the nucleotide will disrupt the interactions between the nucleotide and active-site residues of nucleic acid polymerase and lead to a poor utilization of these modified nucleotides for nucleic acid synthesis by the polymerase.

Unlike most nucleic acid polymerases, the X-family terminal deoxynucleotidyl transferase (Tdt) is a unique class of mesophilic enzyme, which does not rely on a template for adding nucleotides during nucleic acid synthesis. Tdt only requires a short initiator DNA or primer to direct synthesis and incorporation of nucleotides into a growing initiator DNA or primer. Tdt can perform a template-independent DNA synthesis, and the active-site pocket of Tdt is also pre-arranged in a proper geometry for accommodating a canonical nucleotide containing a normal 3′-OH group. Like other nucleic acid polymerases, any substitution of the 3′-OH group with a bulky, chemical group on the nucleotide causes a steric hindrance of nucleotide to fit into the nucleotide-binding pocket of Tdt and results in the reduction of nucleotide-binding affinity and overall DNA synthesis activity of Tdt. Naturally, the template-independent DNA synthesis function of Tdt makes it a primary choice for its application in de novo enzymatic DNA synthesis. However, several intrinsic properties of Tdt, such as limited thermal stability (mesophilic enzyme), preference for certain nucleotide incorporation, intolerance to a larger substitution of the 3′-OH group on the nucleotide and unsatisfactory synthesis efficiency, among others, present a barrier for a practical enzymatic DNA synthesis application.

To broaden the enzymatic DNA synthesis applications, alternative nucleic acid polymerases and their derivatives, which are thermostable and capable of accommodating various non-canonical nucleotide analogues, such as reversible terminator and dye-terminator nucleotides, remain an unmet need.

SUMMARY OF THE INVENTION

Owing to the diverse structure-function relationships mentioned above, the naturally occurring nucleic acid polymerases cannot readily utilize canonical nucleotides and nucleotide analogues as a substrate for de novo nucleic acid synthesis. Thus, the tailor-made, modified nucleic acid polymerase is a prerequisite for exerting the utilities of a variety of nucleic acid synthesis applications.

The inventor has discovered the novel positions/regions in the amino acid sequences of B-family DNA polymerase variants that play crucial parts in endowing the said polymerases with a template-independence and an enhancing nucleotide substrate binding affinity of said polymerases for both canonical and modified nucleotides, thereby improving the nucleic acid synthesis efficiency in the template-independent nucleic acid synthesis method.

Accordingly, in one aspect, the present disclosure provides a B-family DNA polymerase variant comprising: a motif Exo I, a motif Exo II, a motif Exo III, a motif A, a motif B, and a motif C corresponding respectively to the positions 349 to 364, 450 to 476, 590 to 608, 706 to 730, 843 to 855, and 940 to 956 of a consensus sequence (SEQ ID NO:1); a plurality of amino acid substitutions at a position residing in a motif selected from a motif Exo I, a motif Exo II, a motif Exo III, a motif A, a motif B, a motif C, or the combination thereof.

In one embodiment, the B-family DNA polymerase variant is modified from a wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

In one embodiment, the wild-type B-family DNA polymerase is Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetivorans DNA polymerase DNA polymerase (Mac), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), or Bacillus phage Phi29 DNA polymerase (Phi29).

In one embodiment, the B-family DNA polymerase variants provided herein have deficient 3′ to 5′ exonuclease activity.

In one embodiment, the amino acid L or M corresponding to position 715 of SEQ ID NO:1 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y corresponding to position 716 of SEQ ID NO:1 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P of corresponding to position 717 of SEQ ID NO:1 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO:2; and wherein the amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 2 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 410 of SEQ ID NO: 2 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein the amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 2 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 410 of SEQ ID NO: 2 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; and wherein the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; and wherein the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; and wherein the amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 410 of SEQ ID NO: 5 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 411 of SEQ ID NO: 5 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; and wherein the amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 410 of SEQ ID NO: 5 is s not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 411 of SEQ ID NO: 5 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 508 of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein the amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 520 of SEQ ID NO: 9 is not substituted or substituted with A, G, S, or T.

In one embodiment, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein the amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 520 of SEQ ID NO: 9 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In some embodiments, the B-family DNA polymerase variants provided herein exhibits activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogues, or a mixture thereof, to an extendible initiator.

In some embodiments, the extendible initiator comprises a single-stranded oligonucleotide initiator, a blunt-ended double-stranded oligonucleotide initiator, or a mixture thereof.

In some embodiments, the extendible initiator is free form of nucleic acid, in contrast to an immobilized nucleic acid, to be reacted in a liquid phase, such as in liquid medium or other aqueous solutions.

In some embodiments, the extendible initiator is immobilized on a solid support, wherein the solid support comprises a particle, bead, slide, array surface, membrane, flow cell, well, microwell, nano-well, chamber, microfluidic chamber, channel, microfluidic channel, or any other surfaces.

In some embodiments, the at least one nucleotide links with a detectable label.

In some embodiments, the B-family DNA polymerase variant exhibits the activity of incorporating the nucleotide at reaction temperatures ranging from 10° C. to 100° C.

In another aspect, the present disclosure further provides a kit for performing de novo enzymatic nucleic acid synthesis comprising: a B-family DNA polymerase variant derived from a wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, wherein the B-family DNA polymerase variant exhibits activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogues, or a mixture thereof, to an extendible initiator, thereby synthesizing a desired nucleic acid sequence.

Accordingly, the present invention relates to the specific B-family DNA polymerase variants that exhibit an improved performance on incorporating a variety of nucleotides for nucleic acid synthesis at various reaction temperatures in the absence of nucleic acid template. More particularly, the de novo nucleic acid synthesis method can be efficiently performed by said thermotolerant B-family DNA polymerase variants with a broad-spectrum of nucleotides and nucleotide analogues.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will become more readily appreciated with reference to the following description in conjunction with the accompanying drawings.

FIG. 1 shows an amino acid sequence alignment of the wild-type B-family DNA polymerases (PolB) related to the present invention and their consensus sequence.

FIGS. 2A and 2B show the results of the reactions described in Example 3.

FIGS. 3A and 3B show the results of the reactions described in Example 4.

FIG. 4 shows the results of the reactions described in Example 5.1.

FIGS. 5A, 5B, 5C, and 5D show the results of the reactions described in Example 5.2.

FIG. 6 shows the results of the reactions described in Example 5.3.

FIGS. 7A and 7B show the results of the reactions described in Example 5.4.

FIG. 8 shows the results of the reactions described in Example 5.5.

FIG. 9 shows the results of the reactions described in Example 6.

FIG. 10 shows the results of the reactions described in Example 7.1.

FIG. 11 shows the results of the reactions described in Example 7.2.

FIG. 12 shows the results of the reactions described in Example 8.

DETAILED DESCRIPTION OF THE INVENTION Definition

All terms including descriptive or technical terms which are used herein should be construed as having meanings that are understandable to one of ordinary skill in the art. However, the terms may have different meanings according to an intention of the user, case precedents, or the appearance of new technologies. Also, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be described in detail in the descriptions of the present disclosure. Thus, the terms used herein are defined based on the meaning of the terms together with the descriptions throughout the specification. In addition, the titles and subtitles may be attached to the description for readability, but these titles do not affect the scope of the present invention.

As used herein, the term “a,” “an,” or “the” includes plural referents unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably with the term “and/or” unless the context clearly indicates otherwise.

Also, when a part or a method “includes” or “comprises” a component or a step, respectively, unless there is a particular description contrary thereto, the part or the method can further include other components or other steps, not excluding the others.

As used herein, an “amino acid” refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term “amino acid” includes the following twenty natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid or aspartate (Asp or D), cysteine (CyS or C), glutamine (Gln or Q), glutamic acid or glutamate (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). In cases where “X” residues are undefined, these should be defined as “any amino acid”.

The term “functionally equivalent” or “equivalent” is used to describe a specific B-family DNA polymerase (PolB) variant having the substitution or mutation that is considered to occur at the amino acid position in the other PolB, PolB variant according to the sequence alignment, or a reference sequence, which has the same functional or structural role in the enzyme. The equivalent positions may be defined according to homologues, conserved motifs, user-defined, or derived, consensus sequence.

Generally, the homologous PolBs have similar, or identical, amino acid sequences and functional structure, and thereby the equivalent amino acid substitution mutations among different PolBs generally occur at homologous amino acid positions. The term “functionally equivalent” or “equivalent” used herein also encompass mutations that are “homologous” or “positionally equivalent” to a given mutation in view of protein sequence or structural alignment, regardless of the actual function of the mutated amino acid. Practically, the “functionally equivalent”, “homologous” and/or “positionally equivalent” amino acid residues of different polymerases can be identified according to the protein sequence or structural alignment. Accordingly, a cross-species alignment was made on multiple wild-type PolBs, as illustrated in FIG. 1 , and the consensus sequence (SEQ ID NO: 1) is used as a positional reference sequence.

For example, the substitution of amino acid aspartic acid (D) with alanine (A) at position 141 of the wild-type Thermococcus kodakarensis (Kod1) (D141A) amino acid sequence would be functionally equivalent to the amino acid substitution mutation D114A at the conserved residue of wild-type Escherichia phage RB69 DNA polymerase (RB69) amino acid sequence. When the positional reference sequence is used to describe these equivalent amino acid substitutions, the functionally equivalent positions of both amino acid residues 141 of Kod1 and amino acid residue 114 of RB69 corresponds to position 354 of the consensus sequence (SEQ ID NO: 1).

The term “conserved” means the segment of polymerase having the same amino acid residue in the homologous or equivalent position of different PolBs from various sources. The term “semi-conserved” used herein refers to the segment of polymerase that has a similar property of amino acid residue or an identical amino acid residue in the homologous position of different PolBs from various sources.

The terms “nucleic acid”, “nucleic acid sequence”, “oligonucleotide”, “polynucleotide”, and “nucleic acid fragment” as used herein refer to a deoxyribonucleotide or ribonucleotide sequence in a single-stranded or a double-stranded form of which the sources and length are not limited herein; and generally, includes naturally occurring nucleotides or artificial chemical mimics. The term “nucleic acid” as used herein is interchangeable with the terms including natural or unnatural “oligonucleotide”, “polynucleotide”, “DNA”, “RNA”, “gene”, “complementary DNA” (cDNA), and “messenger RNA” (mRNA) in use.

The “nucleic acid”, “oligonucleotide”, or “polynucleotide” used herein refers to a polymer that can be corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymer, or an analogue thereof. This includes polymers of nucleotides such as RNA and DNA, as well as synthetic forms, modified (e.g., chemically or biochemically modified) forms thereof, and mixed polymers (e.g., including both RNA and DNA subunits). Exemplary modifications include methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids and the like). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Typically, the nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids can comprise other linkages (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497-1500, 1991)). A nucleic acid can be or can include, e.g., a chromosome or chromosomal segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, the product of a polymerase chain reaction (PCR), an oligonucleotide, a probe, and a primer. A nucleic acid can be, e.g., single-stranded, double-stranded, or triple-stranded and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence optionally comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.

The nucleic acid as used herein also includes nucleic acid analogue. The term nucleic acid analogue is known to describe compounds or artificial nucleic acids which are functionally or structurally equivalent to naturally existing RNA and DNA. A nucleic acid analogue may have one or more parts of a nucleotide (the phosphate backbone, pentose sugar, and nucleobase) being modified. These modifications on the nucleotide change the structure and geometry of the nucleic acid and its interactions with nucleic acid polymerases. The nucleic acid analogue also encompasses the emerging category of artificial nucleic acids, such as xeno nucleic acids (XNAs), which is designed to have new-to-nature forms of sugar backbone.

Examples of nucleic acid analogues include but are not limited to: the universal bases, such as inosine, 3-nitropyrrole, and 5-nitroindole, which can form a base-pair with all four canonical bases; the phosphate-sugar backbone analogues, such as peptide-nucleic acids (PNA), which affect the backbone properties of the nucleic acid; chemical linker or fluorophore-attached analogues, such as amine-reactive aminoallyl nucleotide, thiol-containing nucleotides, biotin-linked nucleotides, rhodamine-linked nucleotides, and cyanine-linked nucleotides; the fluorescent base analogues, such as 2-aminopurine (2-AP), 3-methylisoxanthopterin (3-MI), 6-methylisoxanthopterin (6-MI), 4-amino-6-methylisoxanthopterin (6-MAP), and 4-dimethylaminopyridine (DMAP); the nucleic acid probes for various genetic applications, such as the oligonucleotide-conjugated with a fluorescent reporter dye (ALEXA, FAM, TET, TAMRA, CY3, CY5, VIC, JOE, HEX, NED, PET, ROX, Texas Red and others) and/or a fluorescent quenchers (BHQs); the molecular beacons (MBs), which are single-stranded nucleic acid probes containing a stem-loop structure and a dual fluorophore-and-quencher label; and the nucleic acid aptamers.

Generally, as used herein, a “template” is a polynucleotide, or a polynucleotide mimic, that contains the desired or unknown target nucleotide sequence. In some instances, the terms “target sequence”, “template polynucleotide”, “target nucleic acid”, “target polynucleotide”, “nucleic acid template”, “template sequence, and variations thereof, are used interchangeably. Specifically, the term “template” refers to a strand of nucleic acid from which a complimentary copy is synthesized from nucleotides or nucleotide analogues through the replication by a template-dependent or template-directed nucleic acid polymerase. Within a nucleic acid duplex, the template strand is, by the convention definition, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand. The “template” strand may also be referred to as the “sense”, or “plus”, strand and the non-template strand as the “antisense”, or “minus”, strand.

The term “initiator” refers to a mononucleoside, a mononucleotide, and oligonucleotide, a polynucleotide, or modified analogues thereof, from which a nucleic acid is to be synthesized by nucleic acid polymerase de novo. The term “initiator” may also refer to a xeno nucleic acids (XNA) or a peptide nucleic acid (PNA) having a 3′-hydroxyl group.

The terms “nucleotide incorporation”, “analogue incorporation”, “incorporating nucleotide” and “incorporating analogue” are known to those skilled in the art and used to describe a process or reaction for nucleic acid synthesis. Thus, as used herein, the term “incorporation” is known to flexibly refer to add one, or more nucleotides, or any specified nucleic acid precursors to the 3′-hydroxyl terminus of a nucleic acid initiator or a primer. For example, the nucleoside triphosphate, such as deoxyguanosine triphosphate (dGTP), is a substrate, or a precursor, for DNA synthesis by DNA polymerase. Once the dGTP is incorporated into the elongated DNA strand, it becomes a deoxyguanosine monophosphate (dGMP) moiety of the newly synthesized DNA. In other words, when a dGTP nucleotide is converted into a dGMP moiety of DNA, the person skilled in the art may say that one dGTP is incorporated into the DNA.

The term “nucleotide analogue” is known to those skilled in the art to describe the chemically modified nucleotides or artificial nucleotides, which are structural mimics of canonical nucleotides. These nucleotide analogues can serve as substrates for nucleic acid polymerases to synthesize nucleic acid. A nucleotide analogue may have one or more altered components of a nucleotide (e.g., the phosphate backbone, pentose sugar, and nucleobase), which changes the structure and configuration of a nucleotide and affects its interactions with other nucleobases and the nucleic acid polymerases. For example, a nucleotide analogue having altered nucleobase may confer alternative base-pairing and base-stacking properties in the DNA or RNA. Furthermore, by way of example, the modification at the base may generate various nucleosides such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other analogues which permits hybridization. In other exemplary aspects, modifications may take place at the level of sugar moiety (for example, replacement of a deoxyribose by an analogue), and/or at the level of the phosphate group (for example, boronate, alkylphosphonate, or phosphorothioate derivatives). A nucleotide analogue monomer may have a phosphate group selected from a monophosphate, a diphosphate, a triphosphate, a tetraphosphate, a pentaphosphate, and a hexaphosphate.

Other examples of nucleotide analogues also include nucleotides having a removable blocking moiety. Examples of the removable blocking moiety include, but are not limited to, a 3′-O-blocking moiety, a base blocking moiety, and a combination thereof. Examples of the 3′-O-blocking moiety include, but are not limited to, O-azido (O—N₃), O-azidomethyl, O-amino, O-allyl, O-phenoxyacetyl, O-methoxyacetyl, O-acetyl, O-(p-toluene)sulfonate, O-phosphate, O-nitrate, O-[4-methoxy]-tetrahydrothiopyranyl, O-tetrahydrothiopyranyl, O-[5-methyl]-tetrahydrofuranyl, O-[2-methyl, 4-methoxy]-tetrahydropyranyl, O-[5-methyl]-tetrahydropyranyl, and O-tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methyl, and O-acyl. Examples of the base blocking moiety may be a reversible dye-terminator. Examples of the reversible dye-terminator include, but are not limited to, a reversible dye-terminator of Illumina MiSeq, a reversible dye-terminator of Illumina HiSeq, a reversible dye-terminator of Illumina Genome Analyzer IIX, a reversible dye-terminator of Helicos Biosciences Heliscope, and a reversible dye-terminator of LaserGen's Lightning Terminators. As used herein, “B-family DNA polymerases (PolBs)” refers to the most common template-dependent nucleic acid polymerases or replicases in all domains of life and many DNA viruses. Like most nucleic acid polymerases, natural PolB s require a duplex primer-template DNA with a free 3′-hydroxyl (3′-OH) group at the primer terminus, all four nucleoside triphosphates (dATP, dTTP, dCTP, and dGTP), and catalytic divalent cations (Mg²⁺ or Mn²⁺, etc.) for catalyzing the nucleotidyl transferase reaction of adding nucleotides to the 3′-OH terminus of a primer. The PolB enzymes, such as bacterial Pol II and archaeal B-family DNA polymerases, are replicative and repair polymerases that inherently contain a catalytic polymerase domain and a 3′ to 5′ exonucleolytic, or proofreading, domain for removing the mis-incorporated nucleotide from the growing primer strand during nucleic acid replication. The term “3′ to 5′ exonucleolytic domain” (Exo domain) refers to a region of the amino acid sequence of a polymerase, which exerts the nucleic acid degradation activity from the 3′-terminus of the primer or the polynucleotide chain. Coordinately, the term “catalytic polymerase domain” (Pol domain) refers to a region of the amino acid sequence of a polymerase, which exerts the catalytic DNA/RNA polymerase activity for adding nucleotides to the 3′-terminus of a primer or a polynucleotide chain.

All known structures of PolB catalytic polymerase domain resemble the shape of human right hand, where the key functional regions are characterized as fingers, palm, and thumb subdomains. The most conserved region is the palm subdomain, which contains the essential residues for catalysis. The protein sequence-alignment among various B-family DNA polymerases from different kingdoms of life and DNA viruses reveals that the PolB polymerases generally harbor six semi-conserved or conserved motifs (I-VI) for their essential exonuclease and polymerase functions. The first three sequence-motifs (Exo I, Exo II, Exo III) are in the Exo domain, while the other three motifs (designated as Motif A, B, and C, respectively) reside in the Pol domain (Hopfner et al, Proc. Natl. Acad. Sci. USA 96, 3600-3605, 1999).

As used herein, the term “mutant” in the context of DNA polymerases of the present invention, means a polypeptide, typically recombinant, that comprises one or more amino acid substitutions relative to a corresponding, functional DNA polymerase.

As used herein, in the context of DNA polymerase variants, “corresponding to another sequence” (e.g., regions, fragments, nucleotide or amino acid positions, or the like) is based on the convention of numbering according to nucleotide or amino acid position number and then aligning the sequences in a manner that maximizes the percentage of sequence identity. An amino acid “corresponding to position X of specific sequence” refers to an amino acid in a polypeptide of interest that aligns with the equivalent amino acid of a specified sequence. Generally, as described herein, the amino acid corresponding to a position of a polymerase can be determined using an alignment algorithm such as BLAST and other currently available tools for conducting amino acid sequence alignment. Because not all positions within a given “corresponding region” need to be identical, non-matching positions within a corresponding region may be regarded or define as “corresponding positions”. Accordingly, as used herein, referral to an “amino acid position corresponding to amino acid position X of a specified DNA polymerase” refers to equivalent positions, based on alignment, in other DNA polymerases and structural homologues and families.

As used herein, the term “consensus sequence of SEQ ID NO: 1” used herein refers to a reference sequence comprising the conserved amino acids of cross-species B-family DNA polymerase. The consensus sequence of SEQ ID NO: 1 is a virtual sequence and is generated by aligned the following 16 wild-type B-family DNA polymerases to obtain the conserved amino acids: Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetivorans DNA polymerase (Mac), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), or Bacillus phage Phi29 DNA polymerase (Phi29). These PolB sequences are aligned for obtaining the alignment sequence as a reference of functionally equivalent positions.

The positions of motifs Exo I, Exo II, Exo III, A, B, and C are defined by the inventor using the consensus sequence of SEQ ID NO: 1 of the present invention; therefore, it shall be noted that the positions of these motifs defined in the present invention are not totally the same as those described in the literature or prior art.

Objectives

The inventor has discovered PolB variants that have an improved function and activity for utilizing canonical nucleotides, nucleotide analogues, and initiators for synthesizing polynucleotides in a template-independent manner. These PolB variants can efficiently add said canonical nucleotides or nucleotide analogues to said initiator in the absence of a replicative template to synthesize a polynucleotide with a random or defined sequence.

More specifically, the inventor has discovered PolB variants can efficiently catalyze the additions of natural nucleotides and nucleotide analogues to the 3′-OH ends of a single-stranded nucleic acid initiator or a blunt-end duplex nucleic acid initiator, in the absence of replicative template, to generate polynucleotides with desired nucleic acid sequences. Furthermore, the PolB variants provided herein generally have a broader substrate specificity, which means the PolB variants can utilize not only naturally occurring nucleotides, but also varieties of modified nucleotides and nucleic acid analogues for the nucleic acid synthesis de novo. Thus, the modified nucleotide can be further designed for being incorporated to the initiator to generate certain functional polynucleotides. Therefore, these PolB variants broaden the scope and utility of template-independent enzymatic nucleic acid synthesis applications for synthesizing polynucleotides with desired sequences and features.

Protein Sequence Alignment of B-Family DNA Polymerases

FIG. 1 shows the amino acid sequence alignment of 16 wild-type B-family DNA polymerases (PolBs) utilized by the inventor, and the outcome of aligned consensus sequence is listed in the bottom (SEQ ID NO:1). The 16 wild-type PolBs being aligned are Thermococcus gorgonarius DNA polymerase (Tgo, SEQ ID NO:2), Thermococcus kodakarensis DNA polymerase (Kod1, SEQ ID NO:3), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N, SEQ ID NO:4), Pyrococcus furiosus DNA polymerase (Pfu, SEQ ID NO:5), Thermococcus litoralis DNA polymerase (Vent, SEQ ID NO:6), Methanosarcina acetivorans DNA polymerase (Mac, SEQ ID NO:7), Pyrobaculum islandicum DNA polymerase, (Pis, SEQ ID NO:8), Sulfolobus solfataricus DNA polymerase (Sso, SEQ ID NO:9), Methanococcus maripaludis DNA polymerase (Mma, SEQ ID NO:10), human DNA polymerase delta catalytic p125 subunit (hPOLD, SEQ ID NO:11), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD, SEQ ID NO:12), Pseudomonas aeruginosa DNA polymerase II (Pae, SEQ ID NO:13), Escherichia coli DNA polymerase II (Eco, SEQ ID NO:14), Escherichia phage RB69 DNA polymerase (RB69, SEQ ID NO:15), Escherichia phage T4 DNA polymerase (T4, SEQ ID NO:16), and Bacillus phage Phi29 DNA polymerase (Phi29, SEQ ID NO:17).

As shown in FIG. 1 , various sequence regions among these exemplary wild-type PolBs are highly conserved while other regions are more variable. Those of skill in the art will immediately recognize and understand that mutations in addition to those specifically identified and discussed herein may be also made in the variable regions of wild-type PolBs without altering, or without substantially altering, the polymerase activity of the mutated enzyme. Likewise, conservative mutations at conserved residues/positions of any of PolBs may be made without altering, or substantially altering, the polymerase activity of the mutated enzyme. Enzyme engineering based on comparative structure analysis with other functionally related enzymes or homologs is a useful technique in the molecular biology field that allows the inventor reasonably predict the effect of a given mutation on the catalytic activity of the enzyme. Based on the present disclosure, using the sequence, the structural data and known physical properties of amino acids, those of skill in the art can mutate enzymes, such as the DNA polymerases encompassed by the present invention, without altering, or without substantially altering, the essential, intrinsic characteristics of the enzymes.

Besides, the motifs Exo I, Exo II, Exo III, A, B, and C corresponding to the positions 349 to 364, 450 to 476, 590 to 608, 706 to 730, 843 to 855, and 940 to 956 respectively of the consensus sequence of SEQ ID NO:1 are focused in the present disclosure. More specifically, the polymerase variant in the present invention is based on substitution mutations at one or more residues correspondingly residing in said motifs.

B-Family DNA Polymerase Variant

In view of the above, provided herein are altered polymerase, which is described based on the amino acid sequence of the consensus sequence of SEQ ID NO: 1. An altered polymerase includes substitution mutations at one or more residues when compared to the consensus sequence of SEQ ID NO: 1. A substitution mutation can be at the same, or homologous, position or a functionally equivalent position compared to the consensus sequence of SEQ ID NO: 1. The skilled person can readily appreciate that an altered polymerase described herein is not naturally occurring. Therefore, an altered polymerase described herein is based on the consensus sequence of SEQ ID NO: 1 and further includes substitution mutations at one or more residues of the corresponding wild-type polymerase (parent polymerase). In one embodiment, at least one substitution mutation is at a position functionally equivalent to an amino acid of the consensus sequence of SEQ ID NO: 1. “Functionally equivalent” means that the altered polymerase has the amino acid substitution at the amino acid position according to the consensus sequence of SEQ ID NO: 1 that has the same functional or structural role in both the consensus sequence and the altered polymerase.

In general, functionally equivalent substitution mutations in two or more different polymerases occur at homologous amino acid positions in the amino acid sequences of the polymerases. Hence, “functionally equivalent” also encompasses mutations that are “positionally equivalent” or “homologous” to a given mutation, regardless of whether or not the particular function of the mutated amino acid is known. It is possible to identify the regions of functionally equivalent and positionally equivalent amino acid residues in the amino acid sequences of two or more different polymerases on the basis of sequence alignment and/or molecular modeling. For instance, the amino acid sequence alignment of exemplary 16 wild-type B-family DNA polymerases from different domains of life are used to identify positionally equivalent and/or functionally equivalent residues. The result of the protein sequence alignment among these PolBs is set forth in FIG. 1 . Thus, for the exemplary residue 141 of the Tgo, Kod1, 9°N, Pfu, and Vent polymerases, residue 171 of the Pis, residue 231 of the Sso, and residue 198 of the Mac polymerase are functionally equivalent and positionally equivalent. Likewise, for the exemplary residue 143 of the Tgo, Kod1, 9°N, Pfu, and Vent polymerases residue 173 of the Pis, residue 233 of the Sso, and residue 200 of the Mac polymerase are functionally equivalent and positionally equivalent. The skilled person can easily identify functionally equivalent residues in DNA polymerases.

In accordance with some embodiments, the provided B-family DNA polymerase variant comprising: a motif Exo I, a motif Exo II, a motif Exo III, a motif A, a motif B, and a motif C corresponding, respectively, to the positions 349 to 364, 450 to 476, 590 to 608, 706 to 730, 843 to 855, and 940 to 956 of the consensus sequence of SEQ ID NO:1; at least one amino acid substitution (one or more amino acid substitutions, or a combination of amino acid substitutions) at positions residing in the motif Exo I, the motif Exo II, and the motif Exo III; and at least one amino acid substitution (one or more amino acid substitutions, or a combination of amino acid substitutions), at positions residing in the motif A, the motif B, and the motif C.

In accordance with the object of the invention to provide a PolB variant having the amino acid sequence as set forth in SEQ ID NO: 1 and the functionally or positionally equivalent sequences, with any substitution or combination of amino acid substitutions listed in Table 1, where the “Essential substitutions” include the substitution motifs, by itself, to confer the activity of de novo nucleic acid synthesis to said PolB variant; and the “Reinforceable substitutions” include the substitution residues that may also confer said activity by itself with a minor capability. Thus, preferably, the “Essential substitutions” can be used alone or in combination with any other mutations, and the “Reinforceable substitutions” are optionally used in combination with the “Essential substitutions” or any other mutations.

TABLE 1 List of PolB variants having the amino acid substitutions according to SEQ ID NO: 1. Name Essential substitutions Reinforceable substitutions YD001 L715A + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD002 L715F + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD003 L715H + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD004 L715I + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD005 L715Q + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD006 L715S + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD007 L715W + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD008 L715Y + Y716A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD009 L715A + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD010 L715F + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD011 L715H + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD012 L715I + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD013 L715Q + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD014 L715S + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD015 L715W + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD016 L715Y + Y716C + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD017 L715A + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD018 L715F + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD019 L715H + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD020 L715I + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD021 L715Q + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD022 L715S + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD023 L715W + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD024 L715Y + Y716D + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD025 L715A + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD026 L715F + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD027 L715H + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD028 L715I + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD029 L715Q + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD030 L715S + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD031 L715W + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD032 L715Y + Y716F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD033 L715A + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD034 L715F + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD035 L715H + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD036 L715I + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD037 L715Q + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD038 L715S + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD039 L715W + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD040 L715Y + Y716G + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD041 L715A + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD042 L715F + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD043 L715H + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD044 L715I + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD045 L715Q + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD046 L715S + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD047 L715W + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD048 L715Y + Y716H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD049 L715A + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD050 L715F + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD051 L715H + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD052 L715I + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD053 L715Q + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD054 L715S + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD055 L715W + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD056 L715Y + Y716I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD057 L715A + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD058 L715F + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD059 L715H + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD060 L715I + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD061 L715Q + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD062 L715S + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD063 L715W + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD064 L715Y + Y716K + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD065 L715A + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD066 L715F + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD067 L715H + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD068 L715I + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD069 L715Q + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD070 L715S + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD071 L715W + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD072 L715Y + Y716L + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD073 L715A + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD074 L715F + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD075 L715H + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD076 L715I + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD077 L715Q + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD078 L715S + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD079 L715W + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD080 L715Y + Y716M + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD081 L715A + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD082 L715F + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD083 L715H + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD084 L715I + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD085 L715Q + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD086 L715S + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD087 L715W + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD088 L715Y + Y716N + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD089 L715A + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD090 L715F + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD091 L715H + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD092 L715I + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD093 L715Q + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD094 L715S + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD095 L715W + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD096 L715Y + Y716Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD097 L715A + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD098 L715F + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD099 L715H + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD100 L715I + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD101 L715Q + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD102 L715S + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD103 L715W + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD104 L715Y + Y716A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD105 L715A + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD106 L715F + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD107 L715H + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD108 L715I + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD109 L715Q + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD110 L715S + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD111 L715W + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD112 L715Y + Y716C + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD113 L715A + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD114 L715F + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD115 L715H + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD116 L715I + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD117 L715Q + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD118 L715S + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD119 L715W + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD120 L715Y + Y716D + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD121 L715A + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD122 L715F + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD123 L715H + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD124 L715I + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD125 L715Q + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD126 L715S + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD127 L715W + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD128 L715Y + Y716F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD129 L715A + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD130 L715F + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD131 L715H + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD132 L715I + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD133 L715Q + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD134 L715S + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD135 L715W + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD136 L715Y + Y716G + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD137 L715A + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD138 L715F + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD139 L715H + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD140 L715I + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD141 L715Q + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD142 L715S + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD143 L715W + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD144 L715Y + Y716H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD145 L715A + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD146 L715F + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD147 L715H + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD148 L715I + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD149 L715Q + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD150 L715S + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD151 L715W + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD152 L715Y + Y716I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD153 L715A + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD154 L715F + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD155 L715H + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD156 L715I + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD157 L715Q + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD158 L715S + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD159 L715W + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD160 L715Y + Y716K + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD161 L715A + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD162 L715F + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD163 L715H + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD164 L715I + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD165 L715Q + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD166 L715S + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD167 L715W + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD168 L715Y + Y716L + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD169 L715A + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD170 L715F + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD171 L715H + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD172 L715I + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD173 L715Q + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD174 L715S + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD175 L715W + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD176 L715Y + Y716M + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD177 L715A + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD178 L715F + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD179 L715H + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD180 L715I + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD181 L715Q + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD182 L715S + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD183 L715W + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD184 L715Y + Y716N + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD185 L715A + Y716Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD186 L715F + Y716Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD187 L715H + Y716Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD188 L715I + Y716Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD189 L715Q + Y716Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD190 L715S + Y716Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD191 L715W + Y716Q + P717G 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A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD232 L715Y + Y716G + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD233 L715A + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD234 L715F + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD235 L715H + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD236 L715I + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD237 L715Q + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD238 L715S + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD239 L715W + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD240 L715Y + Y716H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD241 L715A + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD242 L715F + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD243 L715H + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD244 L715I + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD245 L715Q + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD246 L715S + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD247 L715W + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD248 L715Y + Y716I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD249 L715A + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD250 L715F + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD251 L715H + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD252 L715I + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD253 L715Q + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD254 L715S + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD255 L715W + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD256 L715Y + Y716K + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD257 L715A + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD258 L715F + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD259 L715H + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD260 L715I + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD261 L715Q + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD262 L715S + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD263 L715W + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD264 L715Y + Y716L + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD265 L715A + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD266 L715F + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD267 L715H + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD268 L715I + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD269 L715Q + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD270 L715S + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD271 L715W + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD272 L715Y + Y716M + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD273 L715A + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD274 L715F + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD275 L715H + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD276 L715I + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD277 L715Q + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD278 L715S + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD279 L715W + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD280 L715Y + Y716N + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD281 L715A + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD282 L715F + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD283 L715H + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD284 L715I + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD285 L715Q + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD286 L715S + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD287 L715W + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD288 L715Y + Y716Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD289 L715A + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD290 L715F + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD291 L715H + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD292 L715I + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD293 L715Q + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD294 L715S + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD295 L715W + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD296 L715Y + Y716A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD297 L715A + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD298 L715F + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD299 L715H + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD300 L715I + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD301 L715Q + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD302 L715S + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD303 L715W + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD304 L715Y + Y716C + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD305 L715A + Y716D + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD306 L715F + Y716D + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD307 L715H + Y716D + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD308 L715I + Y716D + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD309 L715Q + Y716D + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD310 L715S + Y716D + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD311 L715W + Y716D + P717T 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A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD322 L715F + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD323 L715H + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD324 L715I + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD325 L715Q + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD326 L715S + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD327 L715W + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD328 L715Y + Y716G + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD329 L715A + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD330 L715F + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD331 L715H + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD332 L715I + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD333 L715Q + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD334 L715S + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD335 L715W + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD336 L715Y + Y716H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD337 L715A + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD338 L715F + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD339 L715H + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD340 L715I + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD341 L715Q + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD342 L715S + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD343 L715W + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD344 L715Y + Y716I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD345 L715A + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD346 L715F + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD347 L715H + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD348 L715I + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD349 L715Q + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD350 L715S + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD351 L715W + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD352 L715Y + Y716K + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD353 L715A + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD354 L715F + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD355 L715H + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD356 L715I + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD357 L715Q + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD358 L715S + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD359 L715W + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD360 L715Y + Y716L + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD361 L715A + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD362 L715F + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD363 L715H + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD364 L715I + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD365 L715Q + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD366 L715S + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD367 L715W + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD368 L715Y + Y716M + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD369 L715A + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD370 L715F + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD371 L715H + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD372 L715I + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD373 L715Q + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD374 L715S + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD375 L715W + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD376 L715Y + Y716N + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD377 L715A + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD378 L715F + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD379 L715H + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD380 L715I + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD381 L715Q + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD382 L715S + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD383 L715W + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD384 L715Y + Y716Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD385 L715A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD386 L715F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD387 L715H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD388 L715I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD389 L715Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD390 L715S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD391 L715W A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD392 L715Y A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD393 L715A + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD394 L715F + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD395 L715H + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD396 L715I + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD397 L715Q + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD398 L715S + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD399 L715W + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD400 L715Y + Y716A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD401 L715A + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD402 L715F + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD403 L715H + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD404 L715I + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD405 L715Q + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD406 L715S + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD407 L715W + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD408 L715Y + Y716C A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD409 L715A + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD410 L715F + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD411 L715H + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD412 L715I + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD413 L715Q + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD414 L715S + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD415 L715W + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD416 L715Y + Y716D A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD417 L715A + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD418 L715F + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD419 L715H + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD420 L715I + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD421 L715Q + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD422 L715S + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD423 L715W + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD424 L715Y + Y716F A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD425 L715A + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD426 L715F + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD427 L715H + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD428 L715I + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD429 L715Q + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD430 L715S + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD431 L715W + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD432 L715Y + Y716G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD433 L715A + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD434 L715F + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD435 L715H + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD436 L715I + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD437 L715Q + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD438 L715S + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD439 L715W + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD440 L715Y + Y716H A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD441 L715A + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD442 L715F + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD443 L715H + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD444 L715I + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD445 L715Q + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD446 L715S + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD447 L715W + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD448 L715Y + Y716I A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD449 L715A + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD450 L715F + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD451 L715H + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD452 L715I + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD453 L715Q + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD454 L715S + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD455 L715W + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD456 L715Y + Y716K A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD457 L715A + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD458 L715F + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD459 L715H + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD460 L715I + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD461 L715Q + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD462 L715S + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD463 L715W + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD464 L715Y + Y716L A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD465 L715A + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD466 L715F + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD467 L715H + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD468 L715I + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD469 L715Q + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD470 L715S + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD471 L715W + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD472 L715Y + Y716M A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD473 L715A + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD474 L715F + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD475 L715H + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD476 L715I + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD477 L715Q + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD478 L715S + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD479 L715W + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD480 L715Y + Y716N A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD481 L715A + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD482 L715F + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD483 L715H + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD484 L715I + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD485 L715Q + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD486 L715S + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD487 L715W + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD488 L715Y + Y716Q A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD489 L715A + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD490 L715F + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD491 L715H + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD492 L715I + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD493 L715Q + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD494 L715S + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD495 L715W + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD496 L715Y + P717A A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD497 L715A + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD498 L715F + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD499 L715H + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD500 L715I + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD501 L715Q + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD502 L715S + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD503 L715W + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD504 L715Y + P717G A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD505 L715A + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD506 L715F + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD507 L715H + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD508 L715I + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD509 L715Q + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD510 L715S + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD511 L715W + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD512 L715Y + P717S A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD513 L715A + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD514 L715F + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD515 L715H + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD516 L715I + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD517 L715Q + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD518 L715S + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD519 L715W + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y YD520 L715Y + P717T A854C/A854D/A854E/A854F/A854H/A854G/A854L/A854R/A854T/A854K/A854Y

In accordance with some embodiments, the B-family DNA polymerase variant is modified from a wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17, which are derived from the wild-type B-family DNA polymerase of Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanosarcina acetivorans DNA polymerase (Mac), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), Methanococcus maripaludis DNA polymerase (Mma), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (SecPOLD), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), and Bacillus phage Phi29 DNA polymerase (Phi29), respectively.

In accordance with some embodiments, the polymerases substantially lack a 3′-exonuclease or other editing activities; therefore, the PolB variants provided herein have deficient 3′ to 5′ exonuclease activity. The 3′ to 5′ exonuclease activity deficiency can be reached by any means. For example, practically, the 3′ to 5′ exonuclease activity can be reduced, attenuated, removed or inactivated by modifying the 3′ to 5′ exonucleolytic domain of the polymerase to generate a polymerase that is deficient in or absence of the 3′ to 5′ exonuclease activity. Preferably, the means of amino acid substitution is adapted to modify the 3′ to 5′ exonucleolytic domain. For example, the PolB variants may have functionally equivalent or positionally equivalent substitutions of the native D with A at position 354 (D354A) and the native E with A at position 356 (E356A) in the motif Exo I of SEQ ID NO:1, thereby causing a 3′-5′ exonuclease deficiency.

In accordance with certain embodiments, the amino acid L or M corresponding to position 715 of SEQ ID NO:1 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y corresponding to position 716 of SEQ ID NO:1 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P of corresponding to position 717 of SEQ ID NO:1 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO:2; and wherein the amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 2 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 410 of SEQ ID NO: 2 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein the amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 2 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 410 of SEQ ID NO: 2 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; and wherein the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; and wherein the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having inactive 3′ to 5′ exonuclease activity is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; and wherein the amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 410 of SEQ ID NO: 5 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 411 of SEQ ID NO: 5 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; and wherein the amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 410 of SEQ ID NO: 5 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 411 of SEQ ID NO: 5 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 508 of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (S so) having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein the amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and the amino acid P at position 520 of SEQ ID NO: 9 is not substituted or substituted with A, G, S, or T.

In accordance with some embodiments, the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein the amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; the amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; the amino acid P at position 520 of SEQ ID NO: 9 is not substituted or substituted with A, G, S, or T; and the amino acid A at position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

In accordance with some embodiments, the B-family DNA polymerase variant exhibits activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogues, or a mixture thereof, to an extendible initiator.

In certain embodiments, the extendible initiator comprises a single-stranded oligonucleotide initiator, a blunt ended double-stranded oligonucleotide initiator, or a mixture thereof. In certain embodiments, the extendible initiator is a free form nucleic acid and can be reacted in a liquid phase.

In certain embodiments, the extendible initiator is immobilized on a solid support, wherein the solid support comprises a particle, bead, slide, array surface, membrane, flow cell, well, microwell, nano-well, chamber, microfluidic chamber, channel, microfluidic channel, or any other surfaces.

In certain embodiments, the at least one nucleotide is linked with a detectable label, such as fluorophores, enzymes, radioactive phosphates, digoxygenin, or biotin.

In accordance with some embodiments, the B-family DNA polymerase variant exhibits the template-independent nucleic acid synthesis activity at reaction temperatures ranging from 10° C. to 100° C. For example, the reaction temperature is between 10° C. and 20° C., between 20° C. and 30° C., between 30° C. and 40° C., between 40° C. and 50° C., between 50° C. and 60° C., between 60° C. and 70° C., between 70° C. and 80° C., between 80° C. and 90° C., between 90° C. and 95° C., between 95° C. and 100° C., or any reaction temperatures within a range defined by an upper limit of 15° C., 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., or 100° C. and an lower limit of 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95° C.

Creation of Polymerase Variants

Various types of mutagenesis techniques are optionally used in the present disclosure to modify polymerases to create the variants of the subject application, for instance, using random or semi-random mutational approaches. In general, any available mutagenesis procedure can be used for making polymerase mutants. Such mutagenesis procedures optionally include selection of altered nucleic acids and polypeptides for one or more activity of interest. Procedures that can be used include, but are not limited to: the site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling and combinatorial overlap PCR), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gaped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, degenerate PCR, double-strand break repair, and many others known to skilled person.

Kit for Performing Template-Independent Nucleic Acid Synthesis Reaction

The present invention also provides a kit that includes the PolB variant described herein, for performing de novo enzymatic nucleic acid synthesis reaction, which comprises: a B-family DNA polymerase variant as described above, wherein the PolB variant exhibits activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogues, or a mixture thereof, to an extendible initiator, thereby synthesizing a desired or predetermined nucleic acid sequence.

Optionally, other reagents such as buffers and solutions required for the PolB variant and nucleotide solution are also included. Instructions for use of the assembled or packaged components are also typically but not necessarily included.

Uses of B-Family DNA Polymerase Variant

In some embodiments, the PolB variants described herein can be used to add natural nucleotides or 3′-modified nucleotide analogues to the 3′-hydroxyl (3′-OH) terminus of a single-stranded or a blunt-end, duplex nucleic acids initiator in a template-independent synthesis manner to produce polynucleotides with desired or predetermined sequences.

In some embodiments, the PolB variants described herein can be used to add nucleotides or nucleotide analogues to the 3′-OH termini of arrays of clustered single-stranded or a blunt-end, duplex nucleic acids initiators, which are immobilized or physically confined, and separated on a solid support as described previously; and preferably, the solid support is made of glass and implemented in the form of silicon wafer. Thus, a multiplexing, parallel de novo nucleic acid synthesis can be performed to synthesize large numbers of various polynucleotides or nucleic acids with distinct sequences.

The PolB variant-based method of large-scale, parallel de novo enzymatic nucleic acid synthesis can drive down the overall costs of de novo nucleic acids synthesis, while shortening the time for manufacturing oligonucleotides, synthetic gene constructs, or genomes for emerging bio-economical applications, such as the nucleic acid-based molecular diagnostics, vaccine and pharmaceutical product development, genome editing, synthetic biology applications, and DNA-based digital data storage. In certain embodiments, the PolB variants described herein can be used to add natural nucleotides or 3′-modified nucleotide analogues to the 3′-OH terminus of the extendible initiator or polynucleotide chain in a template-independent synthesis manner to generate polynucleotides with desired sequences.

In certain embodiments, the PolB variants described herein can be used to incorporate the nucleotide conjugates (one of the types of nucleotide analogue defined previously) covalently linked with an enzyme, an antibody, a chemical moiety/group, such as a biotin, a desthiobiotin, or a fluorophore on the base, phosphate moiety, or pentose sugar of nucleotide, to the 3′-terminus of the nucleic acid initiator in a template-independent synthesis manner.

The incorporation of these nucleotide analogues into the nucleic acids by PolB variants during the nucleic acid synthesis concurrently add the desired component, such as an enzyme, an antibody, or a chemical moiety/group to the newly synthesized nucleic acids in a base-specific, site-specific, or sequence-specific manner. Common components used to label or generate nucleic acid probes and conjugates are known in the art, which include, but are not limited to, radiolabeled nucleotides and nucleotide analogues, modified linkers, such as a biotin, a thiol, an azido, or an amine group, fluorophores, enzymes, and antibodies.

Alternatively, in other embodiments, to label or generate nucleic acid probes, the post-synthetic modifications of nucleic acids can be achieved by covalently or non-covalently coupling with an enzyme, an antibody, a chemical moiety/group, or a fluorophore via a modified linker on the base, the phosphate moiety, or the pentose sugar of synthesis nucleotide. As a result, the desired component can be covalently or non-covalently associated with the specific base or connected to newly synthesized nucleic acids.

In some embodiments, the PolB variant-dependent incorporation of linker-modified nucleotide analogues may be used to facilitate the newly synthesized polynucleotides or nucleic acids to be attached, immobilized or physically confined on various solid surfaces, for broadening the application of de novo enzymatic nucleic acid synthesis. Retrospectively, in other embodiments, the newly synthesized sequence-specific nucleic acids with unique labels, tags, or fluorophores can be used in various nucleic acid-based molecular detections, which include, but are not limited to, the fluorescence in situ hybridization (FISH), TaqMan real-time PCR (RT-PCR), real-time fluorescence ligase chain reaction (RT-LCR), real-time fluorescence recombinase-polymerase amplification (RPA) assay, and real-time fluorescence loop-mediated isothermal amplification assay.

EXAMPLES Example 1: Preparation of PolB Variants

The gene synthesis approach and mutagenesis technique are adapted to create exemplary PolB variants according to the properties of conserved/consensus amino acids in the conserved and semi-conserved regions of selective PolBs, which are disclosed herein. For instance, the well-known site-directed mutagenesis approach is conducted to change the amino acid residues in the motif Exo I, motif Exo II, motif Exo III, motif A, motif B, and motif C regions of an exemplary wild-type PolBs listed herein.

In some embodiments, the procedure for obtaining PolB variants is generally divided into three steps, including Step 1: Gene synthesis of wild-type PolB and its 3′ to 5′ exonuclease-deficient (Exo⁻) mutant, Step 2: Construction of the specific PolB variant having the predetermined mutation(s) in the desired region, and Step 3: Expression and purification of wild-type PolB, Exo⁻ mutant, and PolB variant. As described in more detail below, the techniques used in said procedure are well-known to those skilled in the art.

In Step 1, the codon-optimized gene fragment encoding the wild-type, intein-free PolB polymerase is synthesized by Genomics BioSci & Tech Co. (New Taipei City, Taiwan). The 3′ to 5′ exonuclease-deficient (designated as Exo⁻) PolB polymerase is also provided by the same vendor. The superscript “exo⁻” following an abbreviated name of any PolB listed herein means that the designated wild-type PolB has been modified to remove the intrinsic 3′ to 5′ exonuclease activity, which indicates said PolB is a 3′ to 5′ exonuclease-deficient PolB. Preferably, in the Examples of this disclosure, the Exo⁻ means a PolB mutant carrying combinatory mutations at the positions corresponding to D354 of SEQ ID NO: 1, which is substituted with an alanine residue (D354A), and E356 of SEQ ID NO: 1, which is also substituted with an alanine residue (E356A), respectively.

In Step 2, the synthetic wild-type and Exo⁻ PolB genes are respectively subcloned into the pET28b vector using the flanking NdeI and NotI restriction sites. The sequences of recombinant plasmids are confirmed by DNA sequencing. To create the polymerase variant at the desired motif region of the PolB Exo⁻ protein backbone, the site-directed mutagenesis, is conducted. Briefly, the site-directed mutagenesis PCR is performed with the recombinant plasmids using the Q5 Site-directed Mutagenesis Kit from New England Biolabs (Ipswich, Mass.) to introduce the amino acid substitution. The products are first analyzed by 1% agarose gel to confirm the amplicon size and the rest of PCR reaction mixture is then treated with DpnI at 37° C. for an hour. The mixture is further incubated at 70° C. for 10 mins to inactivate the DpnI function. The DpnI-treated PCR reaction mixture is then purified by the Qiagen's QIAquick PCR Purification Kit (Whatman, Mass.). The purified DNA fragment is treated with the mixture of T4 PNK and T4 DNA ligase. The re-circularized PCR-amplified DNA is transformed back into the E. coli cells. The plasmid DNA was later extracted from the E. coli cells using the Qiagen Plasmid Mini Kit (Whatman, Mass.). The mutated sequences of the polymerase variants at the desired motif region, or regions, are confirmed by DNA sequencing.

In Step 3, E. coli Acella cells harboring the plasmid DNA carrying specific polymerase variant gene are grown in 2 L of LB medium supplemented with 0.5% glucose and 50 μg/ml carbenicillin at 37° C. When the cell density reaches an absorbance value at OD600 nm around 0.6˜0.8, an 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to induce protein expression. Cells are grown for additional 4 hours at 37° C. and then harvested by centrifugation at 4° C. for 10 min at 7,000×g. Cell pellets are resuspended with buffer A [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v/v) glycerol] containing 1 mM benzamidine hydrochloride. Cell lysis is achieved by incubation with 50 mg lysozyme on ice for 1 hour followed by sonication. The cell lysate is clarified by centrifugation at 18,000×g for 25 min at 4° C. The clarified crude cell extract is incubated at 70° C. for 30 minutes and then cooled down at 4° C. The heat-treated cell extract is further clarified by centrifugation at 18,000×g for 25 minutes at 4° C. After centrifugation, the supernatant is diluted with buffer A without NaCl and loaded onto a HiTrap Heparin column (Cytiva Life Sciences, Marlborough, Mass., USA) pre-equilibrated in buffer A in the ÄKTA pure chromatography system (Cytiva Life Sciences, Marlborough, Mass., USA). The protein is eluted with the linear 100 mM to 1 M NaCl gradient using the buffer B [50 mM Tris-HCl (pH 8.0), 1 M NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v/v) glycerol]. Column fractions are analyzed by 10% SDS-PAGE. Fractions containing desired protein are pooled and dialyzed against the storage buffer [50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v/v) glycerol] at 4° C. overnight. The dialyzed protein fraction pool containing the target protein is concentrated using an Amicon filter unit (MW cut-off 50,000). The concentrated protein pool is aliquoted and stored at −20° C. Each mutant polymerase variant was purified with the same procedures as described above. The final protein concentration is determined by the Bradford reaction (Bradford, 1976) using the Bio-Rad Protein Assay (Hercules, Calif.) with bovine serum albumin as a standard.

Example 2: Template-Independent DNA Synthesis Assay

The PolB variants provided herein are tested for template-independent DNA synthesis approach. To further evaluate the activities (performance on incorporating naturally occurring nucleotide and nucleotide analogues) of the PolB variants, normal dNTPs or modified nucleotides and a single-stranded DNA initiator or a blunt-end duplex DNA initiator are used herein.

In this example, the following synthetic oligonucleotides are used to determine the template-independent DNA synthesis activity of PolB variants.

FAM-45-mer DNA initiator: 5′-CTCGGCCTGGCACAGGTCCGTTCAGTGCTGCGGCGACCACCGAGG-3′ (SEQ ID NO: 18). This single-stranded oligonucleotide is labeled with a fluorescent fluorescein amidite (FAM) dye at the 5′-end.

Blunt-end duplex DNA initiator: a duplex DNA composed of a 38-mer primer labeled with a fluorescent Cyanine5 (Cy5) dye at the 5′-end (Cy5-38-mer primer) and its complementary 38-mer oligonucleotide (complementary 38-mer DNA). The sequences are listed as below.

Cy5-38-mer primer: (SEQ ID NO: 19) 5′-GCTTGCACAAGTTCGTTCAATGATACGGCGACCACCGA-3′ Complementary 38-mer DNA: (SEQ ID NO.: 20) 5′-TCGGTGGTCGCCGTATCATTGAACGAACTTGTGCAAGC-3′

The blunt-end duplex DNA initiator is formed by annealing the Cy5-38-mer primer with the complementary 38-mer DNA at a molar ratio of 1:1.5 in the 1× TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA] containing 100 mM NaCl. The DNA annealing reaction is performed in the Bio-Rad Thermal Cycler (Hercules, Calif.) by first heating up the sample mixture to 98° C. for 3 minutes and then gradually cooling it down (5° C./30 seconds) to 4° C. The annealed product without overhang is used as the blunt-end duplex DNA initiator.

The template-independent DNA synthesis reaction is performed in the reaction mixtures (10 μl) containing 100 nM of the FAM-45-mer DNA initiator or the blunt-end duplex DNA initiator, 0.25 mM manganese chloride (MnCl₂), and 200 nM of selective PolB variants. The de novo enzymatic DNA synthesis reactions is initiated by the addition of 200 μM of canonical nucleotide mixtures (dNTPs) or nucleotide analogues (such as 3′-O-azidomethyl-dNTP and dye nucleotides). The reactions are allowed to proceed for a certain period (e.g., 5 minutes for conducting the Examples in the following context) and then terminated by adding 10 μl of 2× quench solution (95% de-ionized formamide and 25 mM EDTA) at a predetermined reaction temperature. The sample mixtures are denatured at 95° C. for 10 min and analyzed by 20% polyacrylamide gel electrophoresis containing 8M urea (Urea-PAGE). The de novo enzymatic DNA synthesis reaction products are then visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, Mass., USA).

Alternatively, the template-independent DNA synthesis assay is performed in the reaction mixture (10 μl) containing 50 nM of FAM-45-mer DNA initiator and 200 nM of terminal deoxynucleotidyl transferase (Tdt) obtained from New England BioLabs (Ipswich, Mass., USA) in the Tdt reaction buffer [50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate (pH 7.9), and 0.25 mM CoCl₂]. The de novo enzymatic DNA synthesis reactions is initiated by adding 200 μM of dNTP mixture at various temperatures, ranging from 10° C., 20° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 70° C., 80° C. to 90° C., respectively. Each reaction is allowed to proceed for 30 minutes (or for a certain period such as 5 and 10 minutes) and then stopped by adding 10 μl of 2× quench solution (95% de-ionized formamide and 25 mM EDTA). The sample mixtures are denatured at 95° C. for 10 min and analyzed by 20% polyacrylamide gel electrophoresis containing 8M urea (Urea-PAGE). The de novo enzymatic DNA synthesis reaction products is then visualized by imaging the gel on the Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, Mass., USA).

Example 3: Template-Independent DNA Synthesis Activity of Exonuclease-Deficient PolB Variants

In this example, the Tgo^(exo−), Kod1^(exo−), 9°N^(exo−), Pfu^(exo−), Vent^(exo−), Mac^(exo−), Pis^(exl−), and Sso^(exo−) were selected as exonuclease-deficient enzymes constructed as described in Example 1. For brevity, the Kod1^(exo−), Vent^(exo−), and Pfu^(exo−) were used as exemplary exonuclease-deficient PolB variant for demonstrating the baseline of template-independent enzymatic DNA synthesis activity. The template-independent enzymatic DNA synthesis activity of commercially available Tdt was also evaluated. The procedures for evaluating the template-independent enzymatic DNA synthesis activity were performed as set forth in Example 2.

The assay results are shown in FIGS. 2A and 2B, where “S” stands for using the substrate (FAM-45-mer DNA initiator) as a blank (no enzyme) control. The results showed that the Kod1^(exo−), Vent^(exo−), and Pfu^(exo−) each exerts the template-independent enzymatic DNA synthesis activity using single-strand DNA as the initiator at incremental reaction temperatures (FIG. 2B), while Tdt rapidly loses its activity when the reaction temperature is raised to around 45° C. (FIG. 2A). The results demonstrated that the exonuclease-deficient PolB variants possessed better thermotolerant properties for template-independent nucleic acid synthesis than that of the conventional Tdt enzyme.

Example 4: Template-Independent DNA Synthesis Activity of Exonuclease-Deficient PolB B Variants

In this example, the Kod1^(exo−) and Pfu^(exo−) as described in Example 3 were further used as exemplary exonuclease-deficient PolB variant for demonstrating the catalytic efficiency PolB variants. The catalytic efficiency was evaluated using the same activity assay as described above, and the DNA synthesis activity was monitored over a defined course of time (e.g., 60 minutes). The results are shown in FIGS. 3A and 3B. As shown in FIGS. 3A and 3B, where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control; and “C” stands for the reaction without the addition of dNTPs as a negative control. The newly synthesized DNA was clearly observed after 5-minute reaction, indicating the enzymatic DNA synthesis reactions were efficient. In addition, it was observed that the amount of newly synthesized DNA product accumulated while the FAM-45-mer DNA initiator prominently diminished over time, indicating the enzymatic DNA synthesis reactions were completed within around 30 minutes. Based on these results, it can be concluded that the exonuclease-deficient PolB variants can effectively and efficiently perform a template-independent enzymatic DNA synthesis.

Example 5: Catalytic Activity of PolB Variants on Incorporating Canonical Nucleotides to the FAM-45-mer DNA Initiator

Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolBs, the selected exonuclease-deficient PolBs (e.g., Tgo^(exo−), Kod1^(exo−), 9°N^(exo−), Pfu^(exo−), Vent^(exo−), Mac^(exo−), Pis^(exo−), and Sso^(exo−)) were further modified to include more amino acid substitutions with different amino acids in varied conserved regions or motifs of each protein.

Example 5.1: Template-Independent DNA Synthesis Activity of Sso Variants

In this example, the PolB variants derived from Sso (SEQ ID NO: 9) is used exemplarily for evaluating the template-independent DNA synthesis activity of the PolB variant carrying combinatory substitutions in the motif Exo I, the motif A, and the motif B. Additionally, the U.S. Pat. No. 11,136,564B2 disclosed an AAI motif for substituting the conserved motif of some archaeal DNA polymerases to improve the incorporation of nucleotide analogues for template-dependent DNA synthesis reactions (i.e., DNA sequencing). The conserved motif is functionally and positionally equivalent to the L715, Y716, and P717 residing in the motif A of the consensus sequence (SEQ ID NO: 1) as defined herein. Therefore, the conserved motif is also functionally and positionally equivalent to the L518, Y519, and P520 residing in the motif A of the wild-type Sso (SEQ ID NO: 9). Thus, in view of the effects of the AAI motif on the template-directed nucleotide incorporation, the AAI motif substitution is equivalently included in this example for comparison.

In this example, the variants modified from Sso^(exo−) (S01) backbone exemplified are numbered and listed in Table 5.1. The template-independent enzymatic DNA synthesis activities of these Sso variants are evaluated using the same activity assay as described above. The results are shown in FIG. 4 , where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control. As shown in FIG. 4 , the variant S02 carrying amino acid substitutions in the motif Exo I (D231A+E233A) and motif A (L518Y+Y519A+P520G) and variant S03 carrying amino acid substitutions in the motif Exo I (D231A+E233A) and motif B (A601L) both exerted prominent catalytic activity of template-independent enzymatic DNA synthesis; and most of the initiator (>95%) substrate was reacted to yield a great quantity of newly synthesized DNA products. Moreover, the variant S05 carrying combinatory amino acid substitutions in the motif Exo I (D231A+E233A), motif A (L518Y+Y519A+P520G), and motif B (A601L) further enhanced the said catalytic activity and produced the longer length of newly synthesized DNA products as compared to the variants S02 and S03. However, the variant S04 carrying combinatory amino acid substitutions in the motif Exo I (D231A+E233A), motif A (L518A+Y519A+P520I) and motif B (A601L) showed a marginal activity.

TABLE 5.1 List of amino acid substitutions in the PolB variants derived from Sso. Type of PolB Variant Enzyme No. Modification Substitutions Sso S01 Exo⁻(D231A + E233A) S02 D231A + E233A + L518Y + Y519A + P520G S03 D231A + E233A + A601L S04 D231A + E233A + L518A + Y519A + P520I + A601L S05 D231A + E233A + L518Y + Y519A + P520G + A601L

Example 5.2: Template-Independent DNA Synthesis Activity of Vent Variants

In this example, the PolB variants derived from Vent (SEQ ID NO: 6) is used exemplarily for evaluating the template-independent DNA synthesis activity of the PolB variant carrying combinatory substitutions in the motif Exo I, the motif A, and the motif B.

In this example, the variants modified from Vent^(exo−) (V01) backbone exemplified are numbered and listed in Table 5.2. The template-independent enzymatic DNA synthesis activities of these Vent variants are evaluated using the same activity assay as described above. The results are shown in FIGS. 5A, 5B, 5C and 5D, where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control. As shown in FIG. 5A, the variant V01 carrying amino acid substitutions in the motif Exo I (D141A+E143A) exerted baseline catalytic activity of template-independent enzymatic DNA synthesis at the reaction temperature of 55° C.; and the variant V03 carrying amino acid substitutions in the motif Exo I (D141A+E143A) and motif B (A488L) exerted an improved DNA synthesis activity as compared to variant V01. Furthermore, as shown in FIG. 5B, the variants V02, V05, and V06 carrying amino acid substitutions in the motif Exo I and motif A (detail substitutions are shown in Table 5.2) also exhibited a robust DNA synthesis activity at the hyperthermal reaction temperature of 70° C. Moreover, as shown in FIG. 5C, the variant V06 and the variant V07 carrying combinatory amino acid substitutions in the motif Exo I, motif A and motif B (detail amino acid substitutions are shown in Table 5.2) also exhibited significant DNA synthesis activity at the hyperthermal reaction temperature of 70° C. Additionally, the variants V08, V09, and V10 (detail amino acid substitutions are shown in Table 5.2) carrying combinatory amino acid substitutions in the motif Exo I, motif A and motif B were also exemplified in this example to demonstrate functional substitutions in the motif Exo I, motif A and motif B as compared to the control enzyme (Tdt). As shown in FIG. 5C, the variants having combinatory substitutions in both motif A and motif B, such as V04, V07, V08, V09, and V10, exhibit superior catalytic activity at wide hyperthermal reaction temperatures (i.e., 60° C. to 90° C.).

TABLE 5.2 List of amino acid substitutions in the PolB variants derived from Vent. Type of PolB Variant Enzyme No. Modification Substitutions Vent V01 Exo⁻(D141A + E143A) V02 D141A + E143A + L411Y + Y412A + P413G V03 D141A + E143A + A488L V04 D141A + E143A + L411Y + Y412A + P413G + A488L V05 D141A + E143A + L411C + Y412A + P413G V06 D141A + E143A + L411V + Y412A + P413G V07 D141A + E143A + L411Y + Y412A + A488L V08 D141A + E143A + L411Q + Y412A + P413G + A488L V09 D141A + E143A + L411Y + Y412A + P413G + A488E V10 D141A + E143A + L411Y + Y412A + P413G + A488F

Example 5.3: Template-Independent DNA Synthesis Activity of 9°N Variants

In this example, the PolB variants derived from 9°N (SEQ ID NO: 4) is used exemplarily for evaluating the template-independent DNA synthesis activity of the PolB variant carrying combinatory substitutions in the motif Exo I and the motif A.

Similarly, the variants modified from 9°N^(exo−) (N01) backbone exemplified are numbered and listed in Table 5.3. The template-independent DNA synthesis activities of these 9°N variants are evaluated using the same activity assay as described above. The results are shown in FIG. 6 , where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control. As shown in FIG. 6 , the variants N02, N03 and N04 carrying amino acid substitutions in the motif Exo I (D141A+E143A) and motif A (detail amino acid substitutions are shown in Table 5.3) exerted robust DNA synthesis activity at the hyperthermal reaction temperature of 70° C.

TABLE 5.3 List of amino acid substitutions in the PolB variants derived from 9° N. Type of PolB Variant Enzyme No. Modification Substitutions 9° N N01 Exo⁻(D141A + E143A) N02 D141A + E143A + L408Y + Y409A + P410G N03 D141A + E143A + L408Y + Y409A + P410T N04 D141A + E143A + L408Y + Y409A + P410V

Example 5.4: Template-Independent DNA Synthesis Activity of Kod1 Variants

In this example, the PolB variants derived from Kod1 (SEQ ID NO: 3) is used exemplarily for evaluating the template-independent DNA synthesis activity of the PolB variant carrying combinatory substitutions in the motif Exo I, the motif A, and the motif B. Additionally, as the rationale described previously in Example 5.1, the AAI motif substitution is also functionally and positionally equivalent to the conserved motif of L715, Y716, and P717 residing in the motif A of the consensus sequence (SEQ ID NO: 1). Therefore, the conserved motif is also functionally and positionally equivalent to the L408, Y409, and P410 residing in the motif A of the wild-type Kod1 (SEQ ID NO: 3); and the AAI motif substitution is equivalently included in this example for comparison.

Similarly, the exemplified variants modified from Kod1^(exo−) (K01) backbone are numbered and listed in Table 5.4. The template-independent enzymatic DNA synthesis activities of these Kod1 variants are evaluated using the same activity assay as described above. The results are shown in FIG. 7A (the synthesis reactions were performed at 55° C.) and 7B (reactions performed at 70° C.). As shown in FIG. 7A, the variant K02, which carries amino acid substitutions in the motif Exo I (D141A+E143A) and motif B (A485L) and the variant K05, which carries amino acid substitutions in the motif Exo I (D141A+E143A) and motif A (L408Y+Y409A+P410G) both exerted a template-independent DNA synthesis activity; and the variant K03 carrying combinatory amino acid substitutions in motif Exo I (D141A+E143A), motif A (L408Y+Y409A+P410G), and motif B (A485L) exhibited an improved DNA synthesis activity as compared to the variants K02 and K05. The variant K04 carrying combinatory amino acid substitutions in the motif Exo I (D141A+E143A), motif A (L408A+Y409A+P4101), and motif B (A485L) showed a lower DNA synthesis activity as compared to the variants K02, K03 and K05. Additionally, as shown in FIG. 7B, where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control, the comparable results for these variants were observed even at the hyperthermal reaction temperature of 70° C.

TABLE 5.4 List of amino acid substitutions in the PolB variants derived from Kod1. Type of PolB Variant Enzyme No. Modification Substitutions Kod1 K01 Exo⁻(D141A + E143A) K02 D141A + E143A + A485L K03 D141A + E143A + L408Y + Y409A + P410G + A485L K04 D141A + E143A + L408A + Y409A + P410I + A485L K05 D141A + E143A + L408Y + Y409A + P410G

Example 5.5: Template-Independent DNA Synthesis Activity of Pfu Variants

In this example, the PolB variants derived from Pfu (SEQ ID NO: 5) is used exemplarily for evaluating the template-independent DNA synthesis activity of the PolB variant carrying combinatory substitutions in the motif Exo I, the motif A, and the motif B. Likewise, as described previously in Example 5.1, the AAI motif substitution is also functionally and positionally equivalent to the conserved motif of L715, Y716, and P717 residing in the motif A of the consensus sequence (SEQ ID NO: 1). Therefore, the conserved motif is also functionally and positionally equivalent to the L409, Y410, and P411 residing in the motif A of the wild-type Kod1 (SEQ ID NO: 5); and the AAI motif substitution is equivalently included in this example for comparison.

Similarly, the variants modified from Pfu^(exo−) (P01) backbone exemplified are numbered and listed in Table 5.5. The template-independent enzymatic DNA synthesis activities of these Pfu variants are evaluated at the reaction temperature of 55° C. using the same activity assay as described above. The results are shown in FIG. 8 . As shown in FIG. 8 , the variant P02 carrying amino acid substitutions in the motif Exo I (D141A+E143A) and motif B (A486L) exerted a template-independent DNA synthesis activity. Furthermore, the variant P03 carrying combinatory amino acid substitutions in motif Exo I (D141A+E143A), motif A (L409Y+Y410A+P411G), and motif B (A486L) exhibited an improved DNA synthesis activity as compared to the variants P02 and P04. The variant P04 carrying combinatory amino acid substitutions in the motif Exo I (D141A+E143A), motif A (L409A+Y410A+P411I; AAI motif), and motif B (A486L) shows a lower DNA synthesis activity as compared to the variants P02 and P03.

TABLE 5.5 List of Amino Acid Substitutions in the PolB variants derived from Pfu. Type of PolB Variant Enzyme No. Modification Substitutions Pfu P01 Exo⁻(D141A + E143A) P02 D141A + E143A + A486L P03 D141A + E143A + L409Y + Y410A + P411G + A486L P04 D141A + E143A + L409A + Y410A + P411I + A486L

Example 5.6: Summary of Template-Independent DNA Synthesis Activity of PolB Variants

In view of above Examples and other comparable template-independent DNA synthesis activity of various PolB variants (data not shown), these results indicate that the amino acid substitutions as provided herein are crucial for conferring or improving the template-independent DNA synthesis activities of the PolB variants.

The functionally or positionally substitutions residing in the motif A and motif B of selected PolB variants are summarized and listed in Table 5.6.

TABLE 5.6 List of amino acid substitutions in the exemplary PolB variants Equivalent substitutions in the motif A and/or motif B corresponding Type of SEQ to the consensus sequence (SEQ ID NO: 1) PolB ID Position 715 Position 716 Position 717 Position 854 Enzymes NO (motif A) (motif A) (motif A) (motif B) Tgo 2 L408A, L408F, Y409A, Y409C, P410A, P410G, A485C, A485D, L408H, L408I, Y409D, Y409F, P410S, P410T A485E, A485F, L408Q, L408S, Y409G, Y409H, A485H, A485G, L408W, L408Y Y409I, Y409K, A485L, A485R, Y409L, Y409M, A485T, A485K, Y409N, Y409Q A485Y Kod1 3 L408A, L408F, Y409A, Y409C, P410A, P410G, A485C, A485D, L408H, L408I, Y409D, Y409F, P410S, P410T A485E, A485F, L408Q, L408S, Y409G, Y409H, A485H, A485G, L408W, L408Y Y409I, Y409K, A485L, A485R, Y409L, Y409M, A485T, A485K, Y409N, Y409Q A485Y 9° N 4 L408A, L408F, Y409A, Y409C, P410A, P410G, A485C, A485D, L408H, L408I, Y409D, Y409F, P410S, P410T A485E, A485F, L408Q, L408S, Y409G, Y409H, A485H, A485G, L408W, L408Y Y409I, Y409K, A485L, A485R, Y409L, Y409M, A485T, A485K, Y409N, Y409Q A485Y Pfu 5 L409A, L409F, Y410A, Y410C, P411A, P411G, A486C, A486D, L409H, L409I, Y410D, Y410F, P411S, P411T A486E, A486F, L409Q, L409S, Y410G, Y410H, A486H, A486G, L409W, L409Y Y410I, Y410K, A486L, A486R, Y410L, Y410M, A486T, A486K, Y410N, Y410Q A486Y Vent 6 L411A, L411F, Y412A, Y412C, P413A, P413G, A488C, A488D, L411H, L411I, Y412D, Y412F, P413S, P413T A488E, A488F, L411Q, L411S, Y412G, Y412H, A488H, A488G, L411W, L411Y Y412I, Y412K, A488L, A488R, Y412L, A488T, A488K, Y412M, A488Y Y412N, Y412Q Mac 7 L485A, L485F, Y486A, Y486C, P487A, P487G, A565C, A565D, L485H, L485I, Y486D, Y486F, P487S, P487T A565E, A565F, L485Q, L485S, Y486G, Y486H, A565H, A565G, L485W, L485Y Y486I, Y486K, A565L, A565R, Y486L, Y486M, A565T, A565K, Y486N, Y486Q A565Y Pis 8 M426A, M426F, Y427A, Y427C, P428A, P428G, A508C, A508D, M426H, M426I, Y427D, Y427F, P428S, P428T A508E, A508F, M426Q, M426S, Y427G, Y427H, A508H, A508G, M426W, M426Y Y427I, Y427K, A508L, A508R, Y427L, A508T, A508K, Y427M, A508Y Y427N, Y427Q Sso 9 L518A, L518F, Y519A, Y519C, P520A, P520G, A601C, A601D, L518H, L518I, Y519D, Y519F, P520S, P520T A601E, A601F, L518Q, L518S, Y519G, Y519H, A601H, A601G, L518W, L518Y Y519I, Y519K, A601L, A601R, Y519L, Y519M, A601T, A601K, Y519N, Y519Q A601Υ

Example 6: Template-Independent DNA Synthesis Activity of PolB Variants on the Blunt-End Duplex DNA Initiator in the Presence of Canonical Nucleotides

Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolBs, the selected exonuclease-deficient PolBs (e.g., Tgo^(exo−), Kod1^(exo−), 9°N^(exo−), Pfu^(exo−), Vent^(exo−), Mac^(exo−), Pis^(exo−), and Sso^(exo−)) were further modified to include additional amino acid substitutions in varied regions of the protein.

For the sake of brevity, in this Example, only the variants modified from Vent^(exo−) (V01) backbone are selected and exemplified. The template-independent DNA synthesis activities of these Vent variants to extend the blunt-end duplex DNA initiator were evaluated using the same activity assay as described above. Only the exemplary results for the representative Vent variants are shown (FIG. 9 ). The list of these variants and their corresponding amino acid substitutions are listed in Table 6.1. The result of gel images for variants V04 and V11-V15 are showed in FIG. 9 . As shown in FIG. 9 , the “S” stands for the substrate (blunt-end duplex DNA initiator) and serves as a blank DNA control, which is also used as a baseline for scoring relative DNA synthesis activity of each variant as described in more details below.

TABLE 6.1 List of amino acid substitutions in the PolB variants derived from Vent. Type of PolB Variant Enzyme No. Modification Substitutions Vent V04 D141A + E143A + L411Y + Y412A + P413G + A488L V11 D141A + E143A + L411Y + Y412C + P413G + A488L V12 D141A + E143A + L411Y + Y412D + P413G + A488L V13 D141A + E143A + L411Y + Y412E + P413G + A488L V14 D141A + E143A + L411Y + Y412F + P413G + A488L V15 D141A + E143A + L411Y + Y412G + P413G + A488L

The relative template-independent DNA synthesis activity of each variant is scored and represented by the number of symbol “+”. The overall activity score for each variant is divided into 4 distinct levels: 1) the “+++” indicates that the initiator is completely converted to various lengths of newly synthesized DNA as compared to the band intensity and position of the substrate control. Hence, the variant is considered to possess an 100% of DNA synthesis activity; 2) the “++” indicates that the initiator is converted around 50% to 100% to various lengths of newly synthesized DNA as compared to the band intensity and position of the substrate control. Hence, the variant is considered to possess a 50% to an 100% of DNA synthesis activity; 3) the “+” indicates that the initiator is converted around 10% to 50% to various lengths of newly synthesized DNA as compared to the band intensity and position of the substrate control. Hence, the variant is considered to possess a 10% to 50% of DNA synthesis activity; and 4) the “+/−” indicates that the initiator is less than 10% converted to various lengths of newly synthesized DNA as compared to the band intensity and position of the substrate control. Therefore, the variant is considered to possess <10% of DNA synthesis activity.

Based on the criteria described above, the activity scoring results of each functional/selected Vent variants are tabulated in Table 6.2.

TABLE 6.2 List of amino acid substitutions and the relative template-independent DNA synthesis activity scoring of PolB variants derived from Vent on blunt-end duplex DNA initiator in the presence of normal dNTPs. Variant No. Residues and Amino Acid Substitutions Activity score V04 D141A + E143A + L411Y + Y412A + P413G + A488L +++ V07 D141A + E143A + L411Y + Y412A + A488L +++ V08 D141A + E143A + L411Q + Y412A + P413G + A488L +++ V11 D141A + E143A + L411Y + Y412C + P413G + A488L ++ V12 D141A + E143A + L411Y + Y412D + P413G + A488L ++ V13 D141A + E143A + L411Y + Y412E + P413G + A488L + V14 D141A + E143A + L411Y + Y412F + P413G + A488L +++ V15 D141A + E143A + L411Y + Y412G + P413G + A488L +++ V16 D141A + E143A + L411C + Y412A + P413G + A488L +++ V17 D141A + E143A + L411A + Y412A + P413G + A488L +++ V18 D141A + E143A + L411F + Y412A + P413G + A488L +++ V19 D141A + E143A + L411H + Y412A + P413G + A488L +++ V20 D141A + E143A + L411I + Y412A + P413G + A488L +/− V21 D141A + E143A + L411S + Y412A + P413G + A488L +++ V22 D141A + E143A + L411T + Y412A + P413G + A488L +++ V23 D141A + E143A + L411W + Y412A + P413G + A488L +++ V24 D141A + E143A + L411Y + Y412H + P413G + A488L +++ V25 D141A + E143A + L411Y + Y412I + P413G + A488L +++ V26 D141A + E143A + L411Y + Y412K + P413G + A488L +++ V27 D141A + E143A + L411Y + Y412L + P413G + A488L +++ V28 D141A + E143A + L411Y + Y412M + P413G + A488L +++ V29 D141A + E143A + L411Y + Y412N + P413G + A488L +++ V30 D141A + E143A + L411Y + Y412Q + P413G + A488L + V31 D141A + E143A + L411Y + Y412R + P413G + A488L + V32 D141A + E143A + L411Y + Y412S + P413G + A488L +++ V33 D141A + E143A + L411Y + Y412T + P413G + A488L +++ V34 D141A + E143A + L411Y + Y412V + P413G + A488L +++ V35 D141A + E143A + L411Y + Y412W + P413G + A488L +++ V36 D141A + E143A + L411Y + P413G + A488L +++ V37 D141A + E143A + L411Y + Y412A + P413A + A488L +++ V38 D141A + E143A + L411Y + Y412A + P413C + A488L +++ V39 D141A + E143A + L411Y + Y412A + P413D + A488L +++ V40 D141A + E143A + L411Y + Y412A + P413E + A488L +++ V41 D141A + E143A + L411Y + Y412A + P413F + A488L +++ V42 D141A + E143A + L411Y + Y412A + P413H + A488L +++ V43 D141A + E143A + L411Y + Y412A + P413I + A488L +++ V44 D141A + E143A + L411Y + Y412A + P413K + A488L +++ V45 D141A + E143A + L411Y + Y412A + P413L + A488L +++ V46 D141A + E143A + L411Y + Y412A + P413M + A488L +++ V47 D141A + E143A + L411Y + Y412A + P413N + A488L +++ V48 D141A + E143A + L411Y + Y412A + P413Q + A488L ++ V49 D141A + E143A + L411Y + Y412A + P413R + A488L + V50 D141A + E143A + L411Y + Y412A + P413S + A488L ++ V51 D141A + E143A + L411Y + Y412A + P413T + A488L ++ V52 D141A + E143A + L411Y + Y412A + P413V + A488L +++ V53 D141A + E143A + L411Y + Y412A + P413W + A488L +++ V54 D141A + E143A + L411Y + Y412A + P413Y + A488L +++

Example 7: Template-Independent DNA Synthesis Activity of PolB Variants on FAM-45-mer DNA Initiator in the Presence of Nucleotide Analogues (3′-O-azidomethyl-dNTPs)

Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolBs, the selected exonuclease-deficient PolBs (e.g., Tgo^(exo−), Kod1^(exo−), 9°N^(exo−), Pfu^(exo−), Vent^(exo−), Mac^(exo−), Pis^(exo−), and Sso^(exo−)) were further modified to include more additional amino acid substitutions in varied protein regions. In this example, exemplary nucleotide analogues 3′-O-azidomethyl-dNTPs (3′-O-AZ-dNTPs), which are well-known reversible terminator nucleotides in the DNA sequencing-by-synthesis chemistry, are used to demonstrate the ability of the PolB variants to utilize non-canonical nucleotides to perform the template-independent nucleic acid synthesis.

Example 7.1: Template-Independent DNA Synthesis Activity of 9°N Variants

For the sake of brevity, in this Example, the variants modified from 9°N^(exo−) (N01) backbone are selected and exemplified. The template-independent DNA synthesis activities of these 9°N variants to extend the FAM-45-mer DNA initiator in the presence of 3′-O-AZ-dCTP are evaluated using the same activity assay as described above. The 9°N derived variants are listed in Table 7.1. Moreover, according to the relative DNA synthesis activity scoring criteria as described in Example 6, the scoring results of each 9°N variants are also illustrated side by side in FIG. 10 , where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control.

As shown in FIG. 10 , the variants N02, N03, and N04 carrying substitutions in the motif Exo I and motif A exhibited around 50% to 95% of DNA synthesis activity in the presence of 3′-O-AZ-dCTP. The addition of 3′-O-AZ-dCMP to the FAM-45-mer DNA generates a 46-mer DNA product.

TABLE 7.1 List of amino acid substitutions in the PolB variants derived from 9° N. Type of PolB Variant Enzyme No. Residues and Amino Acid Substitutions 9° N DNA N05 D141A + E143A + L408Y + P410G + A485V polymerase N06 D141A + E143A + L408Y + Y409A + P410H + A485V N07 D141A + E143A + L408Y + Y409C + P410G + A485V N08 D141A + E143A + L408Y + Y409G + P410G + A485V

Example 7.2: Template-Independent DNA Synthesis Activity of Vent Variants

In this Example, the variants modified from Vent^(exo−) (V01) backbone is selected and exemplified. The template-independent DNA synthesis activities of these Vent variants to extend the FAM-45-mer DNA initiator in the presence of 3′-O-AZ-dATP are evaluated using the same activity assay as described above. The Vent-derived variants are listed in Table 7.2. Moreover, according to the relative DNA synthesis activity scoring criteria as described in Example 6, the relative DNA synthesis activity scoring of each variant are also illustrated in FIG. 11 , where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control.

As shown in FIG. 11 , the variants V04, V17, and V18 carrying combinatory substitutions in the motif Exo I, motif A, and motif B exhibited 100% DNA synthesis activity in the presence of 3′-O-AZ-dATP and produced a 46-mer DNA product.

TABLE 7.2 List of amino acid substitutions in the PolB valiants derived from Vent. Type of PolB Variant Enzyme No. Residues and Amino Acid Substitutions Vent V04 D141A + E143A + L411Y + Y412A + P413G + A488L V16 D141A + E143A + L411C + Y412A + P413G + A488L V17 D141A + E143A + L411A + Y412A + P413G + A488L V18 D141A + E143A + L411F + Y412A + P413G + A488L V19 D141A + E143A + L411H + Y412A + P413G + A488L V22 D141A + E143A + L411T + Y412A + P413G + A488L V23 D141A + E143A + L411W + Y412A + P413G + A488L V55 D141A + E143A + L411G + Y412A + P413G + A488L V56 D141A + E143A + L411V + Y412A + P413G + A488L

In view of above results, a robust template-independent DNA synthesis activity in the presence of 3′-O-AZ-dNTPs were commonly observed on various PolB variants derived from different sources of PolB s provided by the present invention. Accordingly, the Vent variants are further selected to demonstrate the ability of this variants for the template-independent DNA synthesis function. The list of these variants is shown in Table 7.3. The score of relative DNA synthesis activity of each variant in the presence of 3′-O-AZ-dNTP are also shown in Table 7.3 as described above.

TABLE 7.3 List of amino acid substitutions and the relative activity scoring of Vent variants. Variant No. Residue and Amino Acid Substitutions 3′-O-AZ-dNTP Activity score V03 D141A + E143A + A488L 3′-O-AZ-dATP +/− V04 D141A + E143A + L411Y + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V08 D141A + E143A + L411Q + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V09 D141A + E143A + L411Y + Y412A + P413G + A488E 3′-O-AZ-dGTP +++ V10 D141A + E143A + L411Y + Y412A + P413G + A488F 3′-O-AZ-dGTP +++ V11 D141A + E143A + L411Y + Y412C + P413G + A488L 3′-O-AZ-dTTP +/− V12 D141A + E143A + L411Y + Y412D + P413G + A488L 3′-O-AZ-dTTP +/− V14 D141A + E143A + L411Y + Y412F + P413G + A488L 3′-O-AZ-dTTP + V15 D141A + E143A + L411Y + Y412G + P413G + A488L 3′-O-AZ-dTTP +++ V17 D141A + E143A + L411A + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V18 D141A + E143A + L411F + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V19 D141A + E143A + L411H + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V20 D141A + E143A + L411I + Y412A + P413G + A488L 3′-O-AZ-dCTP ++ V21 D141A + E143A + L411S + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V23 D141A + E143A + L411W + Y412A + P413G + A488L 3′-O-AZ-dCTP +++ V24 D141A + E143A + L411Y + Y412H + P413G + A488L 3′-O-AZ-dTTP +/− V25 D141A + E143A + L411Y + Y412I + P413G + A488L 3′-O-AZ-dTTP +/− V26 D141A + E143A + L411Y + Y412K + P413G + A488L 3′-O-AZ-dTTP +/− V27 D141A + E143A + L411Y + Y412L + P413G + A488L 3′-O-AZ-dTTP + V28 D141A + E143A + L411Y + Y412M + P413G + A488L 3′-O-AZ-dTTP + V29 D141A + E143A + L411Y + Y412N + P413G + A488L 3′-O-AZ-dTTP + V30 D141A + E143A + L411Y + Y412Q + P413G + A488L 3′-O-AZ-dTTP +/− V37 D141A + E143A + L411Y + Y412A + P413A + A488L 3′-O-AZ-dCTP +++ V50 D141A + E143A + L411Y + Y412A + P413S + A488L 3′-O-AZ-dCTP +++ V51 D141A + E143A + L411Y + Y412A + P413T + A488L 3′-O-AZ-dCTP ++ V57 D141A + E143A + A488C 3′-O-AZ-dATP +++ V58 D141A + E143A + A488D 3′-O-AZ-dATP +++ V59 D141A + E143A + A488E 3′-O-AZ-dATP +++ V60 D141A + E143A + A488F 3′-O-AZ-dATP +++ V61 D141A + E143A + A488G 3′-O-AZ-dATP +++ V62 D141A + E143A + A488H 3′-O-AZ-dATP +/− V63 D141A + E143A + A488K 3′-O-AZ-dATP +/− V64 D141A + E143A + A488R 3′-O-AZ-dATP +/− V65 D141A + E143A + A488T 3′-O-AZ-dATP +++ V66 D141A + E143A + A488Y 3′-O-AZ-dATP +/− V67 D141A + E143A + L411Y + Y412A + P413G + A488C 3′-O-AZ-dGTP ++ V68 D141A + E143A + L411Y + Y412A + P413G + A488D 3′-O-AZ-dGTP +++ V69 D141A + E143A + L411Y + Y412A + P413G + A488G 3′-O-AZ-dGTP ++ V70 D141A + E143A + L411Y + Y412A + P413G + A488H 3′-O-AZ-dGTP +++ V71 D141A + E143A + L411Y + Y412A + P413G + A488K 3′-O-AZ-dGTP +++ V72 D141A + E143A + L411Y + Y412A + P413G + A488R 3′-O-AZ-dGTP ++ V73 D141A + E143A + L411Y + Y412A + P413G + A488T 3′-O-AZ-dGTP +++ V74 D141A + E143A + L411Y + Y412A + P413G + A488Y 3′-O-AZ-dGTP +++

Example 7.3: Template-Independent DNA Synthesis Activity of Pfu, Kod1, and Sso Variants

In this Example, the variants modified from Pfu^(exo−) (P01), Kod1^(exo−) (K01), and Sso^(exo−) (S01) backbone are selected and exemplified. The template-independent DNA synthesis activities of these variants to extend the FAM-45-mer DNA initiator in the presence of 3′-O-AZ-dATP are evaluated using the same activity assay as described above. The list of variants used in this example and their relative DNA synthesis activity thereof are listed in Table 7.4.

The results show that Pfu^(exo−), Kod1^(exo−), and Sso^(exo−) variants carrying combinatory substitutions exhibited 100% DNA synthesis activity in the presence of 3′-O-AZ-dATP. However, the variant K04 carrying combinatory amino acid substitutions in the motif Exo I (D141A+E143A), motif A (L408A+Y409A+P410I) and motif B (A485L) showed a marginal activity as compared to K03.

TABLE 7.4 List of amino acid substitutions and relative activity scores of Pfu^(exo−), Kod1^(exo−), and Sso^(exo−) variants. Variant Activity No. Type of PolB with Amino Acid Substitutions score P03 Pfu^(exo−) + L409Y + Y410A + P411G + A486L ++ K03 Kod1^(exo−) + L408Y + Y409A + P410G + A485L ++ K04 Kod1^(exo−) + L408A + Y409A + P410I + A485L + S05 Sso^(exo−) + L518Y + Y519A + P520G + A601L ++

Example 8: Template-Independent DNA Synthesis Activity of PolB Variants on FAM-45-mer DNA Initiator in the Presence of Dye-Labeled Nucleotide Analogues

In this Example, the ability of PolB variants to utilize a dye-labeled nucleotide analogue for the template-independent DNA synthesis is further demonstrated by the exemplary PolB variants. The variants modified from Vent^(exo−) (V01) backbone are selected and exemplified. The template-independent DNA synthesis activities of these Vent variants to extend the FAM-45-mer DNA initiator in the presence of a Cy5-labeled dTTP (Cy5-dTTP) are evaluated using the same activity assay as described above. The DNA synthesis activity assay is conducted at the hyperthermal reaction temperature of 70° C. Meanwhile, the Tdt enzyme was also used for direct comparison. The activity assay for Tdt enzyme was conducted at the reaction temperature of 37° C., which is the standard working temperature for the Tdt enzyme. For the sake of brevity, the variant V04 was selected to demonstrate the template-independent DNA synthesis activity in the presence of Cy5-dTTP. The results are shown in FIG. 12 , where “S” stands for the substrate (FAM-45-mer DNA initiator) and serves as a blank DNA control. As shown in FIG. 12 , the variant V04 shows a robust DNA synthesis activity by incorporating Cy5-dTTP to the FAM-45-mer DNA initiator.

In view of the results observed above, the PolB variants and the kit provided herein have been further proven to incorporate a variety of nucleotides effectively and efficiently for de novo enzymatic nucleic acid synthesis; and they are also proven to successfully exert the conferred template-independent DNA synthesis function under broader reaction temperatures covering from atmospheric temperatures to the hyperthermal conditions, demonstrating a higher thermotolerance. Therefore, the PolB variants and the kit within the scope of the present disclosure can broaden the scope of various applications of template-independent enzymatic nucleic acids synthesis in different reaction conditions.

The present disclosure has been described with embodiments thereof, and it is understood that various modifications, without departing from the scope of the present disclosure, are in accordance with the embodiments of the present disclosure. Hence, the embodiments described are intended to cover the modifications within the scope of the present disclosure, rather than to limit the present disclosure. The scope of the claims therefore should be accorded the broadest interpretation so as to encompass all such modifications. 

What is claimed is:
 1. AB-family DNA polymerase variant having an amino acid sequence positioned and numbered according to a consensus sequence (SEQ ID NO: 1), comprising a plurality of amino acid substitution mutations at positions residing in a motif selected from a motif Exo I, a motif Exo II, a motif Exo III, a motif A, a motif B, a motif C, or the combination thereof; wherein the motif Exo I, the motif Exo II, the motif Exo III, the motif A, the motif B, and the motif C correspond respectively to positions 349 to 364, 450 to 476, 590 to 608, 706 to 730, 843 to 855, and 940 to 956 of the consensus sequence.
 2. The B-family DNA polymerase variant of claim 1, wherein the B-family DNA polymerase variant is modified from a wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and
 17. 3. The B-family DNA polymerase variant of claim 2, wherein the wild-type B-family DNA polymerase is Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetivorans DNA polymerase (Mac), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), or Bacillus phage Phi29 DNA polymerase (Phi29).
 4. The B-family DNA polymerase variant of claim 3, wherein the B-family DNA polymerase variant has deficient 3′ to 5′ exonuclease activity.
 5. The B-family DNA polymerase variant of claim 4, wherein i. an amino acid L or M corresponding to position 715 of SEQ ID NO: 1 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y corresponding to position 716 of SEQ ID NO: 1 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P corresponding to position 717 of SEQ ID NO: 1 is not substituted or substituted with A, G, S, or T.
 6. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein i. an amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 2 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 410 of SEQ ID NO: 2 is not substituted or substituted with A, G, S, or T.
 7. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein i. an amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 2 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 410 of SEQ ID NO: 2 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 8. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein: i. an amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 410 of SEQ ID NO: 3 is not substituted or substituted with A, G, S, or T.
 9. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein i. an amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 410 of SEQ ID NO: 3 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 10. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; and wherein i. an amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 410 of SEQ ID NO: 4 is not substituted or substituted with A, G, S, or T.
 11. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; and wherein i. an amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 410 of SEQ ID NO: 4 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 12. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; and wherein i. an amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 410 of SEQ ID NO: 5 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 411 of SEQ ID NO: 5 is not substituted or substituted with A, G, S, or T.
 13. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; and wherein i. an amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 410 of SEQ ID NO: 5 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 411 of SEQ ID NO: 5 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 14. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein i. an amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 413 of SEQ ID NO: 6 is not substituted or substituted with A, G, S, or T.
 15. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein i. an amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 413 of SEQ ID NO: 6 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 16. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein i. an amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 487 of SEQ ID NO: 7 is not substituted or substituted with A, G, S, or T.
 17. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein i. an amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 487 of SEQ ID NO: 7 is not substituted or substituted with A, G, S, or T; and iv. the amino acid A at position 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 18. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein i. an amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 428 of SEQ ID NO: 8 is not substituted or substituted with A, G, S, or T.
 19. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein i. an amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 428 of SEQ ID NO: 8 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 508 of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 20. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein i. an amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and iii. an amino acid P at position 520 of SEQ ID NO: 9 is not substituted or substituted with A, G, S, or T.
 21. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant having deficient 3′ to 5′ exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein i. an amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; ii. an amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; iii. an amino acid P at position 520 of SEQ ID NO: 9 is not substituted or substituted with A, G, S, or T; and iv. an amino acid A at position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.
 22. The B-family DNA polymerase variant of claim 5, wherein the B-family DNA polymerase variant exhibits an activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotide, nucleotide analogue, or a mixture thereof, to an extendible initiator.
 23. The B-family DNA polymerase variant of claim 22, wherein the extendible initiator comprises a single-stranded oligonucleotide initiator, a blunt-ended double-stranded oligonucleotide initiator, or a mixture thereof.
 24. The B-family DNA polymerase variant of claim 22, wherein the extendible initiator is free form nucleic acid to be reacted in a liquid phase.
 25. The B-family DNA polymerase variant of claim 22, wherein the extendible initiator is immobilized on a solid support, wherein the solid support comprises a particle, bead, slide, array surface, membrane, flow cell, well, microwell, nano-well, chamber, microfluidic chamber, channel, microfluidic channel, or any other surfaces.
 26. The B-family DNA polymerase variant of claim 22, wherein the at least one nucleotide is linked with a detectable label.
 27. The B-family DNA polymerase variant of claim 22, wherein the B-family DNA polymerase variant exhibits the activity at reaction temperatures from 10° C. to 100° C.
 28. A kit for performing de novo enzymatic nucleic acid synthesis, comprising a B-family DNA polymerase variant derived from a wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, wherein the B-family DNA polymerase variant exhibits activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotide, nucleotide analogue, or a mixture thereof, to an extendible initiator, thereby synthesizing a desired nucleic acid sequence. 